Category: College of Engineering

Research in Mechanical Engineering

Mixed Mode Fracture Characterization of Hydrogel-3D Printed Polymer Adhesively Bonded Systems 

By Nicholas Sardinha

Introduction 

Hydrogels are soft polymer networks with an interstitial liquid, usually water, and they have many applications ranging from tissue engineering, water purification [1], and even anti- biofouling. However, for hydrogels to be more useful for anti-biofouling, their adhesion to different surfaces must be investigated. Many marine vessels have hulls made of different metals such as steel and aluminum, but with the rise of additive manufacturing came the increase in the use of 3D-printed polymers as hull materials. This research attempted to study the adhesive bonding of hydrogel and 3D-printed acrylonitrile butadiene styrene (ABS). The specific method chosen to evaluate the fracture toughness was the J-integral method, used on a mixed mode bending fixture described in the methodology section of this report and based on the work of Zhao Y, Seah LK, Chai GB [2]. Mixed mode bending applies opening mode-I and shearing mode-II to the double cantilever beam (DCB) specimen. However, this type of test has never been conducted with two materials with such a large difference in elastic moduli, with hydrogel having a Young’s modulus in the 10 kPa – 2 MPa range and ABS in the 2-4 GPa range. This causes the ABS to experience much less deformation than the hydrogel at the same load. That is the primary reason why the traditional peel test was not used, as hydrogel experiences significant deformation, which absorbs energy. This leads to the peel test not accurately measuring fracture toughness of softer materials, since that additional energy absorption is not properly accounted for in fracture toughness calculations. 

Methodology 

Specimen Production 

Table 1: Hydrogel Composition 

Number of Samples  Total Solution (ml)  DI Water (ml)  AAm (g)  DAC (ml)  MBAA 

(mg) 

 KA (mg) 
1  25  14.8  8.9  10.2  23.2  13.7 
3  75  44.4  26.7  30.6  69.6  41.1 

The specimens used in this research were fabricated using a unique in situ fabrication method. Firstly, a hydrogel precursor solution was made using the five main components: deionized water (DI Water), Acrylamide (AAm) monomer, Acryloyloxyethyltrimethyl ammonium chloride (DAC) cationic monomer, N,N’-Methylenebisacrylamide (MBAA) crosslinker, and α-Ketoglutaric acid (KA) photoinitiator. From Table 1 the weight percentage of AAm and DAC are 25.24 wt.% and 32.69 wt.%, respectively. This process produces a clear hydrogel precursor solution that can be used for the fabrication of test specimens. 

The second half of each specimen is a 3D-printed ABS T-shape, which was produced using a Stratasys uPrint 3D printer and ABS P430. After the ABS T-shape finished printing, a 10 mm long pre-crack film was glued to the top surface, as shown in the red area of Figure 1(a). 

 For the control samples, the rest of the 3D-printed surface was left unaltered, and the ABS T- shape was placed into a silicone mold. This silicone mold was designed with two connected cavities: one for the ABS and the other for the hydrogel precursor solution. The side for the precursor solution had an opening that was sealed using a strip of photocopier film and two glass microscope slides. The purpose of using transparent materials to seal that side of the mold is to ensure that the 365 nm ultraviolet light can pass through and photocure the hydrogel. After many experiments, a 4-hour photocuring time was chosen and used for all samples. Since the mold had an opening on the top for the hydrogel precursor solution to be poured in, a sheet of plastic wrap was applied around the entire mold to prevent moisture from entering or exiting during curing. Photocopier film was also used to line the inside of the mold to ensure that the surface of the hydrogel would be smoother. 

After the samples finished curing, they were removed from the mold, and excess hydrogel was cut away with a razor blade so that the edge of the hydrogel aligned with the edge of the ABS. Once this was completed, a 20× 100 mm2 strip of photocopier film was superglued to the backside of the hydrogel. Next, a 100 mm long strip of paper with millimeter markings was applied to the side of the ABS to help measure the pre-crack length. To ensure that the pre- crack area did not have any adhesion, the crack was slightly opened by hand, and the end of the pre-crack was marked on the side of the ABS with a black marker. 

Photooxidation 

The photooxidation process refers to the exposure of the ABS surface to chlorine dioxide (ClO2) gas and 365 nm ultraviolet light. To produce ClO2, 100 mg of sodium chlorite (NaClO2) was added to 14 ml of DI water inside a small dish. This small dish was then transferred into a larger glass reaction vessel. Any ABS samples to be photooxidized were then placed in the reaction vessel close to the small dish. The entire reaction vessel was then placed in the fume hood. Then using an adjustable pipette, 100 µl of 37% hydrochloric acid (HCl) was added to the solution, which starts producing chlorine dioxide gas. After that, the reaction vessel was sealed using plastic wrap and a clear flat plastic sheet to contain all the produced gas. Then the ultraviolet light was placed on top of the reaction vessel, and a timer was started as soon as the light was switched on. After the desired time was completed, the ABS samples were transferred into another container, where they could be used immediately for either contact angle measurements or for fabrication of mixed mode bending specimens. All remaining reacted solution was then transferred into a designated waste container specifically for that solution. 

Mixed Mode Bending Fixture 

Figure 1:(a) Hydrogel/ABS adhesive system and (b) T-Shape drawing with dimensions in millimeters. 

 

Figure 1(a) shows the ABS/hydrogel adhesive systems, and Figure 1(b) shows the dimensions of the T-shape part of either portion. The double T-shape specimens were placed into a mixed-mode bending fixture as shown below in Figure 2. This fixture was loaded under compression with a load P, where force and displacement measurements were recorded using a Shimadzu AGS-X universal testing machine (UTM) with a 50N load cell. A Celestron handheld digital microscope was used to capture the crack initiation. The loads at locations A, B, C, and D were determined using statics, and the angles measured at these four locations were used in determining the fracture toughness. Due to the significant difference in elastic moduli between the hydrogel and ABS, θB & θD are extremely small and approximated as zero. 

 

Figure 2: Mixed mode bending loading configuration of the adhesive system. 

 

The lever length c can be adjusted to change the mode mixity, and for these experiments a length of 30.61 mm was used. The horizontal distance between points B and C called length L in Figure 2 cannot be adjusted and was set to 45 mm. Prior to each set of tests, the load 50 N load cell was calibrated. 

To secure the prepped samples into the fixture, superglue was applied to the top surface of the hydrogel to meet the grip, and the grips were tightened down. The lower grips on the ABS side did not require superglue, since the ABS did not deform during testing. Once the specimen was in the fixture, the pivot was slowly lowered until it contacted the surface, then a vertical line was drawn onto the hydrogel to measure the angle at point C. Both the force and displacement were then zeroed. A loading rate of 4 mm per minute was used based on previous mixed mode tests. The recording on the digital microscope and the UTM were started at the same time and two screenshots were taken, one at the start of the experiment and one at fracture initiation. 

These screenshots were then imported into MATLAB, where a code was used to analyze the angles of 3 different points. The first screenshot was analyzed to measure the initial angle of the vertical line at point C, and the second screenshot was analyzed to measure the angle of the fixture, the angle at A, and the angle at C (this is shown below in Figure 3). The time of fracture initiation was also recorded from this video and was used to get the load and displacement at fracture initiation. With the P, θA, & θC, the fracture toughness can be calculated using the following formula:

= 100( sin() + sin() − sin() + sin() ) 

The average fracture toughness and 95% confidence interval were plotted using Excel. The force vs displacement was also plotted using MATLAB and the Excel spreadsheets produced by the UTM. After the completion of each test, the hydrogel was separated from the ABS and the ABS surface was cleaned using deionized water for future use. This procedure was repeated until four consistent tests were performed. 

Figure 3: Example of Angle Measurements at Fracture Initiation. 

 

Water Contact Angle Measurement 

Water contact angle measurements were taken using a Ramé-Hart Model 90 goniometer. The goniometer was calibrated using the included calibration sphere and the water contact angle software package. The surface to be tested was placed on the test platform with the backlight turned on and leveled using the included adjustment knobs. After that, a droplet of deionized water was dropped on the surface using the included syringe, and the contact angle was recorded. This was repeated in four separate locations on the ABS surface on two different specimens. This procedure was repeated for the three different conditions which were untreated, 15-minute photooxidation, and 30-minute photooxidation. The average and 95% confidence interval were then plotted in Excel. 

Results 

Figure 4: Water Contact Angle for Untreated, 15 Minute Photooxidation, and 30 Minute Photooxidation. 

 

The water contact angle measurements from Figure 4 showed a significant difference between untreated samples and samples that have had the photooxidation procedure done on them. The average contact angle decreased from 76.23° to 42.06°. This was an expected result as the photooxidation process modifies the ABS surface with more reactive groups, meaning the surface should become more hydrophilic, which it did. It also showed that increasing the photooxidation treatment time from 15 minutes to 30 minutes did not have much of an effect. This is important information as it led to one of the challenges experienced later on in the process. 

 

Figure 5: Control Load vs Displacement. 

 

The load versus displacement graph in Figure 5 shows the same trend for all specimens. There is an initial shallower slope section that quickly transitions into a linear portion until the peak load is achieved, and then the load quickly drops off as the crack grows along the interface. All four specimens achieved peak load values within 1 N of each other. 

Figure 6: Control Fracture Initiation Toughness. 

 

The average fracture toughness for the untreated control samples was 31.1 J/m2 as seen in Figure 6. There are no other conditions to compare to the control which is explained in the discussion section of this report. 

Discussion 

Many challenges were faced during this research that limited the scope of this project. Due to the novelty of bonding hydrogel and ABS plastic, a unique hydrogel precursor solution had to be created and iterated on. Initially, the hydrogel used had half of the concentration of AAm and DAC, which caused the hydrogel to be very soft, excessively deform, and ultimately lead to inconclusive tests. This challenge was eventually overcome by doubling the concentration of AAm and DAC, along with increasing the MBAA cross-linker concentration. 

This led to very successful control experiments. However, after the control tests concluded, photooxidation tests started. Based on the contact angle results, a 10-minute photooxidation procedure was selected. As expected, the photooxidation had a major effect on the adhesive bond between the surfaces; it had such a large effect that the hydrogel consistently failed prior to the interfacial bond. Corrective measures were attempted, such as decreasing the concentration of the NaClO2 and HCL solution by half and the exposure time down to 3 minutes. This still resulted in the same challenges as previous tests, namely crack propagation into the hydrogel. 

The second-largest challenge was determining the proper method to measure θC. The methods used by other researchers who used the same fixture did not work as expected due to the ABS acting as a rigid body and causing the hydrogel to indent rather than bend. This indentation issue was compounded by the fact that the hydrogel used in these experiments was prone to warping. Many different solutions were proposed to combat those problems, but ultimately, applying a backing layer to the hydrogel and drawing a vertical line at point C was chosen, as described in the methodology section of this report. 

Conclusion / Future Work 

There are many more aspects of this research topic that can be further investigated, the most pressing being the degree to which the photooxidation process increases the fracture toughness. This would require the control samples to be redone using a hydrogel stronger than an interfacial bond to prevent cracks from propagating into the hydrogel. Once the hydrogel is strong enough to withstand the photo-oxidized tests, other test configurations can be tested, such as modifying the surface geometry or roughness. Following that, another future step for this research is using digital image correlation (DIC) to determine the mode mixity of these mix mode tests. The lever length used for these tests was chosen because, with the same material for top and bottom T-shapes, it produces equal parts mode I & II loading conditions. However, since the top and bottom are not the same material, the mixity is not the same. With the help of DIC, the strain fields near the crack tip can be analyzed to determine the true mode mixity. Overall, this research has built up some knowledge of hydrogel and ABS adhesively bonded systems. Even though it did not achieve all of its goals from its onset, it still provides a solid foundation that can be built upon by future researchers. 

References 

Yahia LH, Chirani N, Gritsch L, et al. History and Applications of Hydrogels. J Biomedical Sci. 2015, 4:2. http://dx.doi.org/10.4172/2254-609X.100013. 

 Y. Zhao, L. K. Seah, G. B. Chai, Measurement of interlaminar fracture properties of composites using the J-integral method, J. Reinf. Plast. Compos. 35 (2016) 1143-1154. https://doi.org/10.1177/0731684416642031. 

 

Research in Chemistry and Biochemistry

Microwave Mediated Synthesis of Quinazolinone Natural products of  Peganum harmala for Medicinal Chemistry Applications 

By Amelie Duval
Introduction

In the pharmaceutical field, quinazolinones have become an important pharmacophoric scaffold due to their presence in natural compounds with a wide range of medicinal chemistry applications. Among these, Peharmaline A (1), a natural alkaloid with b-Caroline and tricyclic pyrroloquinazolinone cores (Figure 1) that exhibits various biological activities, including antimalarial, anticancer, anti-inflammatory, and antibacterial activities has garnered significant interest of synthetic and medicinal chemists in recent years. In addition, quinazolinones possess stability and change adaptability that makes it easy to prepare, which is prime for scientific investigations. Peharmaline A is found in Peganum harmala L., a species coming from the family of medicinally important Zygophyllaceae. In Chinese traditional medicine, P. harmala L. seed extract is widely used to treat malignancies of the digestive system and malaria. 

(±}-Peharmaline A (1} 

Figure 1: (±)-Peharmaline A precursors 

Objective of the Proposal 

We proposed to develop a novel approach to synthesize Peharmaline A utilizing the deoxyvasicinone and 5-methoxytryptamine, and extend the same to access a library of analogues of peharmaline to further study their biomedicinal potential. 

Methodology – for the Synthesis of (±)-Peharmaline A and Its Analogues 

According to our retrosynthetic plan shown in Scheme 1, we have proposed a total synthetic route to achieve (±)-Peharmaline A 1 in a three-step process: through Pictet-Spengler reaction with the use of 5- methoxytryptamine 3 and deoxyvasicinone methyl oxalate intermediate 2, which itself can be made by of the acylation reaction of deoxyvasicinone 4 with methyl oxalyl chloride 5. It required us to develop a short and effective synthesis for deoxyvasicinone itself through adapting and modifying the reported synthesis into a practical large–scale under microwave irradiation chemistry one-pot synthesis. Starting from commercially available isatoic anhydride 6 and pyrrolidinone 7, they would serve as the building blocks and will result in (±)-Peharmaline in just three steps. It would also provide a divergent approach towards the (±)-Peharmaline A analogues in a modular fashion. 

Scheme 1: Retrosynthetic plan of synthesizing (±)-Peharmaline A 

Results and Discussions 

Scheme 1 describes our efforts toward the development of a total synthetic approach to the synthesis of (±) -Peharmaline A. After a lot of careful experimentation with stoichiometry and heating (temperatures and time intervals), we finally optimized the synthesis of deoxyvasicinone to work on 1Og scale. It required thorough mixing of 1:1.25 equals of isatoic anhydride 5 and the pyrrolidinone 6 and heating of three minutes at medium power household MW (with one-minute intervals). Once the reaction was completed, it was allowed to cool to room temperature, and any remaining starting materials were removed under reduced pressure. The next step was acylation of the deoxyvasicinone 4. The acylation reaction conditions needed a lot of optimizations. We started with the available chemical in the lab i.e., ethyl oxalyl chloride 6 instead of methyl oxalyl chloride, which will lead to Methyl (±)-Peharmaline A, while we awaited the other chemical to arrive from the vendor. 

After a great deal of experimentation to optimize the temperature conditions, the equivalence of acylation reaction, and the solvent environment, we found out that 3 eq of ethyl oxalyl chloride and 2 eq of Et3N base in anhydrous DCM was needed to complete the reaction with deoxyvasicinone, resulting in  32.6 % of yield. Further, it was confirmed that the initially maintained O °C temperature needs to be increased after the complete addition (1 h) of ethyl oxalyl chloride to overnight refluxing to 48 h for the completion of the reaction to obtain a green clean solid product. The addition of both ethyl oxalyl chloride and Et3N base was separated into two portions, with the first half equivalent being added in at OoC initially, and the second half equivalent being added after 24 hours of refluxing in the same method before placing the reaction back to reflux for an additional 24 h. The acylated deoxyvasicinone 2 was obtained in enol form and it was confirmed by NMR and X-ray crystallography. 7 The methyl oxalyl chloride had arrived from the vendor and was tested with the same chemical and temperature environment, and experiments are still on-going to examine the identity and quality of the product. Additional tests are being conducted with deoxyvasicinone with benzoyl chloride and oxalyl chloride to determine if analogues can be developed to further the research with the final (±)-Peharmaline A and other quinazolinone products. 

Scheme 2: Synthesis approach of (±)-Peharmaline A analogue 

 

The next step in the sequence was the Pictet-Spengler reaction. We decided to establish it first with the commercially available tryptamine instead of the expensive 5-methoxy tryptamine (required to be synthesized in the lab later). Accordingly, we combined the tryptamine base 3.85b with acylated deoxyvasicinone 2 under refluxing conditions. After a lot of experimentation trials, our efforts towards the Pictet-Spengler reaction conditions were successful. It ensued in the presence of a catalytic amount of acid that could facilitate the reaction forward to the formation of (±)-Peharmaline A analogue when there was no water present in the sample. As we ran these optimization reactions on a mg scale, we obtained the product through preparative TLC thus far. We need to further optimize them on a larger scale and obtain the pure compound. 

 

Scheme 3: Pictet-Spengler reaction for the final assembly 

 

Figure 2 shows the changes in the 1H NMR peaks along the path of (±)-Peharmaline A analogue (8) (C) formation from the deoxyvasicinone 4 (A). 

Figure 2: Comparison of (±)-Peharmaline A analogue synthesis process A) 1H NMR of Deoxyvasicinone 4 in CDCl3 synthesized through MW irradiation chemistry. B) 1H NMR of enol form of acylated deoxyvasicinone 2 in CDCl3) 1H NMR of Peharmaline A analogue 8 in CDCl3 synthesized in TFMS acid/DCM solution. 

Conclusions and Future Directions

In summary, we have established the groundwork needed for the synthesis of peharmaline A and its analogues. Scheme 4 deliniates our approach of making a library of (±)-Peharmaline A analogues by employing different tryptamines and tyrosines–based primary arylethanamines in Pictet-Spengler reaction by applying our optimized reaction conditions (anh. DCM/TFMSA/reflux-24 h) towards appending the b-Carboline ring to the deoxyvasicinone ring. As can be seen, it is a modular and divergent approach to making (±)-Peharmaline A analogues via employing different acid chlorides as well, as seen in Scheme 5. We propose to carry it out when the grad student that I was associated with returns in the next winter and summer breaks. 

 

Scheme 4: Future study: Scoping Pictet-Spengler towards (±)-Peharmaline A analogues 

Scheme 5: Other modifications for Diversity Oriented Synthesis of Peharmaline A 

Acknowledgments

Support from the UMassD OUR is greatly appreciated. Many thanks to Fazmina Anver and Dr. Rasapalli for their teaching and mentoring in the lab. 

References

Kulkarni, A. S.; Dash, A.; Shingare, R. D.; Chand, J.; Manhas, D.; Singh, A.; Nandi, U.; Goswami, A.; Srinivasa Reddy, D. Identification of New Modulator of DNA Repairing Pathways Based on Natural Product   (±)-Peharmaline   A.   Bioorg.   Med.   Chem.   2023,   91,   117365. https://doi.org/1O.1O16/j.bmc.2O23.117365. 

Piemontesi, C.; Wang, Q.; Zhu, J. Enantioselective Synthesis of (+)-Peganumine A. J. Am. Chem. Soc. 2016, 138 (35), 11148-11151. https://doi.org/1O.1O21/jacs.6bO7846. 

K.-B. Wang, S.-G. Li, X.-Y. Huang, et al. (±)-Peharmaline A: a pair of rare B-carboline-vasicinone hybrid alkaloid enantiomers from Peganum harmalaEur J Org Chem, 2O17 (2O17), pp. 1876-1879, 1O.1OO2/ejoc.2O17OO137 

Anver, F; Rasapalli, S. Thesis: Synthetic Studies Towards Biologically Active Heterocyclic Alkaloids and Their Analogues a dissertation in Chemistry and Biochemistry 

Alsibaee, A. M., Al-Yousef, H. M., & Al-Salem, H. S. (2O23). Quinazolinones, the Winning Horse in Drug Discovery. Molecules (Basel, Switzerland), 28(3), 978. https://doi.org/1O.339O/molecules28O3O978 

Cao, R.; Peng, W.; Wang, Z.; Xu, A. B-Carboline Alkaloids: Biochemical and Pharmacological Functions. Curr. Med. Chem. 2007, 14, 479-5OO. https://doi.org/1O.2174/O929867O777994O998. 

Alshehry, R; Rasapalli, S. Synthetic studies toward biologically active quinazolinones : a dissertation in Chemistry and Biochemistry https://umassd.primo.exlibrisgroup.com. 

 

 

Research in Bioengineering

Smart Microparticles for On-Demand Drug Delivery 

By Abid Neron
Abstract 

Drug delivery systems represent a cornerstone of contemporary medical research, particularly in addressing intractable diseases such as cancer and neurological disorders. Current treatment modalities often result in collateral damage to healthy cells, underscoring the need for targeted delivery methods. Microparticles have emerged as promising candidates for precise drug delivery. This study explores the use of PLGA microparticles, fabricated via double emulsion, for sustained vancomycin hydrochloride release. The microparticles were loaded with Indocyanine Green (ICG) for near-infrared–responsive (NIR) drug delivery. Process parameters were optimized for size, morphology, drug loading, and release kinetics. Anti-bacterial tests showed higher drug release and antibacterial efficiency when exposed to NIR light. Chitosan coating halted drug release in the absence of NIR light, demonstrating controlled, on-demand release. This system offers the potential for targeted and efficient drug delivery. 

Introduction

Drug delivery systems are a crucial component of the drug manufacturing process. An effective drug is rendered useless if it fails to reach its target area. Thus, a drug delivery vehicle must be manufactured to ensure that the drug is properly administered. Traditional drug delivery methods, including oral, topical, and injection, are effective, but all have certain disadvantages, namely, poor bioavailability, instantaneous release of the drug, and lack of site-specific targeting1. These disadvantages can be mediated by using higher concentrations or continuous intaking of the specified drug, which steadily uses more drugs for treatment and can cause damage to other areas. Novel drug delivery systems have been at the forefront of bioengineering research, as finding a more effective vehicle for a drug will increase its effectiveness, thus reducing the amount of the drug needed for treatment and potential side effects. 

Microcarriers are currently spearheading novel drug delivery systems. Most notably, much research is being conducted on microparticles. They are spherical particles that are 1 to 100 μm in diameter. Microparticles have been chosen as a suitable drug delivery vector as they can be tailored to the specific task since they are highly customizable. This degree of customization is due to the different fabrication processes and the numerous materials used to manufacture them. Further, multiple materials can be used in conjunction for a higher degree of complexity when desired. Most importantly among the properties of microparticles is their ability to hold drugs inside of them. An effective drug delivery vector must have numerous properties; among them are drug encapsulation, biodegradability, biocompatibility, sustained release, and implementation of surface modifications. Depending on the fabrication, microparticles can have all these properties. 

Background

The fabrication process is crucial for microparticles as it is the process that gives the particles their properties. Thus, when creating a microparticle for drug delivery, deliberate thought is put into the fabrication process and the desired properties. For instance, a microparticle made with a toxic polymer is rendered useless in the field of drug delivery as it will harm the patient. All microparticles made for drug delivery use a biocompatible polymer such as PLGA, Chitosan, Gelatin, and PCL. Further, surface modifications can be implemented to help increase uptake of the drug by the cells or to target certain cells. Targeted drug delivery increases the effectiveness of the treatment. Lack of site targeting can lead to the drug not working effectively or having undesirable consequences. The most infamous treatment that lacks targeting delivery is Chemotherapy. Chemotherapy targets rapidly dividing cells, a property of cancerous cells. However, it is also a property of hair, skin, and gastrointestinal tract cells. This causes multiple side effects that wouldn’t occur if Chemotherapy only targeted the cancerous cells. 

Microparticles are potential candidates for cancer treatment. They can be modified for targeted drug delivery because cancerous cells can be targeted since they express certain proteins and have special receptors. A biocompatible microparticle fabricated with an effective anti-cancer drug and a specific targeting ligand could revolutionize the way cancer is treated. While cancer treatment is a major application for microparticle-mediated drug delivery, other important applications include treatment for neurological disorders, diabetes, and respiratory and inflammatory diseases.

Poly Lactic-co-Glycolic Acid (PLGA) is the most widely used polymer for microparticle fabrication due to its biocompatibility and FDA approval. PLGA is a co-polymer, meaning it is made using two different monomers: Lactic Acid and Glycolic Acid. The ratio of both these monomers affects the properties of PLGA, giving an extra layer of customization in the fabrication process. Among the properties affected by the ratios is the rate of biodegradation, which increases with the increase of Glycolic Acid. In addition, it supports numerous fabrication methods as well as surface modifications and has good drug encapsulation properties. 

Near-infrared (NIR) light, which includes wavelengths from 800 to 2,500 nm, is invisible to the naked eye. Indocyanine Green (ICG) is a photothermal material and thus will react with certain wavelengths of light and convert light energy into heat energy. This central concept was utilized in fabricating the microparticles for targeted drug release, as the use of NIR light in PLGA-ICG particles should accelerate drug release, providing a vessel for on-demand delivery. To further prevent drug elution, chitosan-coated microparticles were fabricated as well. The chitosan should prevent drugs from eluting except when exposed to NIR light due to the photothermal reaction occurring. 

Methods

Microparticles Fabrication: 

The PLGA-ICG-Van. HCl microparticles were fabricated using the double emulsion method as shown in Figure 1, with the external phase being 1% PVA. 

Figure 1: Schematic for Fabrication of PLGA-ICG-Van.HCl microparticles 

 

Drug Release: 

To test for drug release from the MPs, 10 mg of fabricated PLGA MPs containing ICG and Van.HCl was placed into a 2 ml solution of phosphate-buffered saline (PBS) with a pH of 7.4 inside a Cytivia dialysis kit with a cut-off of 8 kDa. The microparticles were exposed to near-infrared (NIR) light at a wavelength of 808nm. The experiment set up is shown in Figure 2. Sample absorbance values were recorded, and a vancomycin hydrochloride standard curve was used to determine the drug’s concentration. 

Figure 2: Drug Release Set Up 

 

Anti-Bacterial Activity: 

Escherichia coli S17 (E. coli) was cultured in LB broth and incubated overnight at 37°C at 250 RPM for 24 hours. Fresh bacterial cultures were prepared for each antibacterial test using the previously refreshed culture. For the antibacterial test, a new batch of bacteria was cultured overnight until reaching an optical density at 600 nm (OD600) of 0.5, corresponding to approximately 8 × 10^8 cells/ml. In a microcentrifuge tube, 10 mg of microparticles were combined with 1 ml of LB broth and inoculated with 10 µl of the bacterial culture. Each microparticle group was tested in duplicate: one sample was exposed to NIR light at 1 W for 30 minutes, while the other sample was not exposed to NIR light. To ensure consistency, the unexposed sample was kept at room temperature for 30 minutes. Both samples were then incubated at 37°C for 2 hours. After incubation, 100 µl of each sample was spread onto an LB agar plate and incubated at 37°C for 24 hours to allow visualization of bacterial growth where bacterial colonies were counted. A control group prepared using the same protocol but without microparticles, was included for comparison. 

Results 

I: Microparticle Fabrication: 

Four different microparticle concentrations were fabricated: PVI2, PVI4, PVI5, and PVI6. Each variation had a different concentration of ICG. ICG concentrations can be found in Table 1. 225 mg of PLGA and 75 mg were used for the fabrication of all the microparticles. PVI4 was chosen to be coated with chitosan as it was found to have the highest drug release (Figure 3) and the highest antibacterial activity (Figure 4). 

Table 1: ICG Concentrations in Microparticle Variations 

Variation  ICG Concentration (mg) 
PVI2  3.875 
PVI4  38.75 
PVI5  19.375 
PVI6  7.75 

 

II. Drug Release:

Vancomycin hydrochloride release for each microparticle variation with and without exposure to NIR light can be seen in Figure 3. NIR light-exposed samples consistently have a higher drug release. The PVI4 exposed to NIR light had the highest drug release, followed by the PVI4 sample not exposed to NIR light. 

Figure 3: Vancomycin Hydrochloride Release for Each Microparticle Variation 

 

III. Antibacterial Activity: 

Antibacterial activity, when subjected to microparticles, is utilized as a visualization of the microparticles’ drug release and to further verify the drug release results. It is used as an in vitro test for fabricated microparticles. Antibacterial activity is shown by the number of colonies present on the LB agar plate. The resulting colony counts are found in Figure 4. Chitosan-coated particles’ antibacterial activity can be seen in Figure 5. 

Figure 4: Bacterial Colonies Counted for each Microparticle Variation 

Figure 5: Bacterial Colonies Counted for Chitosan Coated PVI4 

Discussion 

The microparticles exhibited good drug encapsulation and drug release with varying results depending on the specific variation used. Fabrication of four variations of the microparticles with differing ICG content demonstrated the effects of ICG in drug release when subjected to NIR light. PVI4 had the highest drug release and antibacterial activity as the ICG accelerated the release and deterioration of the PLGA microparticles. The uncoated samples released vancomycin hydrochloride almost instantly when subjected to solution. To mitigate undesired drug release, chitosan-coated microparticles released less vancomycin hydrochloride when not exposed to NIR light. Meanwhile, when exposed to NIR light, the vancomycin hydrochloride was released in a larger concentration. The concentration of drug release is less than its uncoated counterpart (PVI4). Chitosan-coated PLGA – ICG microparticles are potentially an excellent candidate for targeted and on-demand drug release as the drug inside is trapped and released when subjected to NIR light. Both drug release and antibacterial activity further emphasize the potential applications of PLGA – ICG microparticles in drug delivery. 

Future Directions

Further research into the usage of unstable drugs such as alkylating agents must be undertaken. Cancer drugs are unstable and further modifications to the microparticles might be needed for the PLGA to hold these drugs. Moreover, further in vitro testing with mammalian cell lines is needed. Finally, specific cell targeting surface modifications on the microparticles will enhance the targeting of the microparticles to the desired cell. 

Acknowledgements 

I would like to thank Mr. Mishal Pokharel, a BMEBT Ph.D. student; without his guidance and expertise, this project wouldn’t have existed. He invited me into his lab and allowed me to assist with his Ph.D. project, for which these results were gathered for. I’d also like to thank Dr. Tracie Ferreira for being an extraordinary supervisor and always being open to my questions and new ideas. Finally, I’d like to thank the Office of Undergraduate Research and the Honors College for their generous support and for believing in me and my project. Their funding allowed the continuation of this project.

 

 

 

Research in Bioengineering

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Suicide Vectors for allelic exchange in Cellulophaga Lytica

By Mehul Puri

Introduction 

C. Lytica 

The gram-negative marine-based bacteria C. lytica has a genome of 3,765,936 base pairs, including 3,303 protein-coding genes and 55 RNA genes (Pati et al., 2011). It can grow in a wide range of temperatures between 4 °C and 40 °C in an 8% NaCl concentration with optimal growth between 22 °C to 30 °C (Pati et al., 2011). In the absence of flagella and pili, C. lytica cells depend on gliding motility to transport themselves. This translocation mechanism is also used to form biofilm colonies, which consist of colonies of C. lytica cells that can grow on non- biological surfaces such as rocks and metals. This allows them to survive in hostile environments and colonize new environments with ease. (Hall-Stoodley et al., 2004). 

Additionally, biofilm colonies serve as a foundation for larvae growth due to chemical and physical cues (Unabia et al., 1999). The formation of biofilm colonies by C. lytica also produces iridescence or coloration created by light reflection on intricately organized cells resembling crystals, as shown in Figure 1 (DeSimone, 2021). Though this iridescence has not been observed in natural environments, it has been observed in colonies grown in lab environments, and the significance of this iridescence in nature has not been elucidated to date. It is one of the identifiers or markers of biofilm colonies for C. lytica cells (Kientz et al., 2016). 

Figure 1. Colonies of C. lytica grown on Black ink plates (Adapted from M. DeSimone’s thesis, DeSimone 2021) 

Goal 

This study hypothesizes that deletion of the GldB gene in the bacteria Cellulophaga Lytica is responsible for gliding motility and can disrupt the formation of colonies. The ability to disrupt the formation of uniform colonies of C. lytica can impact the biofilm formation and allow us to control the iridescence of the bacteria. 

Approach/Methodology 

Designing the Vector 

In this project, the PYT313 suicide vector (Donated by a collaborator’s lab, Dr. Yontao Zhu, Minnesota State University Mankato) was used as it works with F. johnsoniae related to C. lytica. The suicide vector, as shown in Figure 2, contains sacB and the promoter of F. johnsoniae, ompA, which is used to construct chromosomal gene deletions specific to gliding (Zhu, 2017). Additionally, PYT313 is resistant to the antibiotic ampicillin due to the presence of AmpR. 

Figure 2. The plasmid map of PYT313 donated by Dr. Yongtao Zhu indicating the presence of the sacB, erythromycin resistance (ermF), and the promoter of F. johnsoniae, ompA (Zhu et al., 2017). 

Four primers are designed to isolate the GldB (gliding motility) gene within the C. Lytica DNA and are then used to create a new suicide vector using PYT313. As shown in figure 3, primers a and d contain restriction enzyme sites on their 3’ and 5’ sites, respectively. These sites correspond to specified restriction enzyme sites on the PYT313 vector. Primers c and d are homologous 1 kb upstream and downstream of the GldB gene from the start and stop codons, respectively. Through three polymerase chain reactions (PCR), the AB fragment and CD fragment are used to create the AD fragment which contains the GldB gene with restriction enzyme sites upstream and downstream of the DNA (Francis et al.). 

 

Figure 3. Four primers are designed for Overlap PCR. Through two PCR rounds, the gene is removed from the bacteria C. Lytica and ligated onto the PYT313 suicide vector. Figure from (Francis et al.). 

Then, through double digestion, the PYT313 vector is digested at the two specified restriction enzyme sites. After running the gel purification through electrophoresis, the larger digested PYT313 DNA is extracted and ligated with the AD fragment containing the GldB gene. This creates a new vector specifically designed to replace the GldB gene within C. Lytica with an inactive copy of the gene through transformation and conjugation processes (Francis et al.). 

Transformation and Conjugation 

Bacterial transformation is the process of environmental DNA uptake by competent cells. In this project, chemically competent E. Coli S17 λ Pir cells are used to uptake the GldB gene-inclusive PYT313 suicide vector. S17 cells allow for better DNA transfer during conjugation, which is why DNA uptake during transformation is crucial for GldB gene deletion. (Chen et al.) Then, the transformed E. Coli S17 cells are conjugated with C. Lytica cells for a direct transfer of DNA. 

Bacterial conjugation directly transfers genetic material from the E. Coli S17 λ Pir cells to the C. Lytica. During the conjugation, the mutant GldB gene is introduced to the recipient C. Lytica. As shown in figure 4, a two-step homologous recombinant event occurs: first and second crossover. 

During the first crossover, C. Lytica acquires the plasmid from the S17 cells, including the ampicillin antibiotic resistance. The conjugated bacteria is isolated using antibiotics, and a second crossover event occurs using the SacB sucrose counter-selection gene. During this event, the remaining part of the vector is removed from the C. Lytica, including the ampicillin resistance, leaving behind the mutant GldB gene or a wild-type GldB gene. Colony PCR is then conducted to differentiate between the two outcomes. C. Lytica cells with mutant GldB gene are grown and tested for results (Old Reliable: Two-Step Allelic Exchange by Bitesize Bio). 

Figure 4. Two Step Homologous Recombinant Event occurs, creating two types of bacteria. First conjugated C. Lytica has a wild-type allele, and second has the desired mutant allele. Adapted from Old Reliable: Two-Step Allelic Exchange by Bitesize Bio. 

Projected Outcome 

To test if the combination of DNA transfer is successful, the conjugated C. Lytica cells are introduced to antibiotic ampicillin. If the cells survive, the conjugation was successful, otherwise the cells would deteriorate. Additionally, successful conjugation will result in the C. Lytica cells losing their ability of iridescence. 

Results 

Spring 2024 

During the Spring 2024 semester, getting results on the transformation and conjugation was emphasized rather than altering the PYT313 to create a new vector. C. Lytica and PYT313 were grown in BB2 Agar and LB Agar plates respectively, as shown in Figure 5. Afterwards, a 50 ml culture was made using the colonies from both plates. Additionally, a 50 ml culture of S17 λ pir cells was grown for transformation. Transformation protocol was conducted using the S17 cells and PYT313. Transforming the S17 λ pir cells using the PYT313 was successful as it resulted in colony growth on LB Agar plates. Colony growth on multiple plates with antibiotic present is shown in Figure 6.


Figure 5. Growth of C. Lytica and PYT313 in BB2 Agar and LB Agar plates. LB Agar plate has ampicillin antibiotic added to it which demonstrates PYT313 ampicillin resistance. 

Figure 6. LB Agar plates with PYT313 suicide vector transformed S17 λ pir cells. Growth shows successful transformation due to the presence of antibiotic ampicillin. 

However, conjugation was unsuccessful, as no growth was present in the Conjugation Plates after a week of incubating. This could be due to multiple factors such as too many antibiotics or less cell density of C. Lytica or transformed S17 cells before conjugation. Additionally, due to time constraints, multiple trials of transformation and conjugation could not be completed. 

Summer 2024 

During Summer 2024, our aim is to establish which primers to use for the PYT313 for the two- step allelic exchange and complete successful transformation and conjugation with the new vector. Because we know transformation is possible, and conjugation can also be achieved with multiple trials, we hope to achieve complete deletion/replacement of the GldB by the end of summer. Additionally, the OUR grants have greatly enhanced this project by providing funds for resources and have made my research goals possible.

References 

ChenInês, et al. “The Ins and Outs of DNA Transfer in Bacteria.” Science, vol. 310, no. 5753, 2 Dec. 2005, pp. 1456–1460, www.ncbi.nlm.nih.gov/pmc/articles/PMC3919525/, https://doi.org/10.1126/science.1114021. 

DeSimone, Mark, Development of Genetic Engineering Tools for the Iridescent Bacteria Cellulophaga lytica, A Thesis (2021) 

 Francis, Matthew S, et al. “Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components.” Methods in Molecular Biology, 12 Nov. 2016, pp. 11–31, pubmed.ncbi.nlm.nih.gov/27837478/, https://doi.org/10.1007/978-1-4939-6649-3_2. 

Accessed 13 June 2024. 

Hall-Stoodley, L., Costerton, J. W., & Stoodley, P. (2004). Nature Reviews Microbiology, 2(2), 95–108. https://doi.org/10.1038/nrmicro821 

Kientz, B. et al. A unique self-organization of bacterial sub-communities creates iridescence in Cellulophaga lytica colony biofilms. Sci. Rep. 6, 19906; doi: 10.1038/srep19906 (2016). 

McBride, M. J., & Zhu, Y. (2013). Gliding Motility and Por Secretion System Genes Are Widespread among Members of the Phylum Bacteroidetes. Journal of Bacteriology, 195(2), 270–278. https://doi.org/10.1128/jb.01962-12 

“Old Reliable: Two-Step Allelic Exchange.” Bitesize Bio, 17 July 2018, bitesizebio.com/41461/old-reliable-two-step-allelic- exchange/#:~:text=The%20idea%20behind%20suicide%20vectors,understandably%2C%20can’t %20replicate. 

Pati, A., Abt, B., Teshima, H., Nolan, M., Lapidus, A., Lucas, S., Hammon, N., Deshpande, S., Cheng, J.-F., Tapia, R., Han, C., Goodwin, L., Pitluck, S., Liolios, K., Pagani, I., Mavromatis, K., Ovchinikova, G., Chen, A., Palaniappan, K., & Land, M. (2011). Complete genome sequence of Cellulophaga lytica type strain (LIM-21T). Standards in Genomic Sciences, 4(2), 221–232. https://doi.org/10.4056/sigs.1774329

Unabia, C., Hadfield, M. Role of bacteria in larval settlement and metamorphosis of the polychaete Hydroides elegans. Marine Biology 133, 55–64 (1999). https://doi.org/10.1007/s002270050442

Research in Bioengineering

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Examining the Effect of Vitamin D on Melanoma

 By Mary Goodrow

 

Introduction 

Over the summer and during the spring semester, I was introduced to research and working with live animals. I also learned to keep a lab notebook and record lab activities during this introductory process. The first thing I did was separate fertilized fish eggs from unfertilized ones. First, it was hard to distinguish fertilized eggs from unfertilized ones. However, it was cool to see under the microscope. Later, after the semester had ended, I started learning how to make agarose plates. Agarose plates are needed in the process of producing Zebrafish with melanoma, which is done via recombinant DNA technology. It was a fun learning process being introduced to the lab equipment and procedures for using it. One of the graduate students was nice enough to offer some help along the way. I made some mistakes, especially since I am not used to the sterile technique. I made the mistake of placing the lids of the plates on the counter. It’s now ingrained in me to open the top of the lid only slightly when working with Petri dishes.  

Methods 

In my lab notebook, I kept track of all the instructions given to me by Dr. Ferreira, mainly the ones directly related to the Agarose plates. Each page was formatted based on the date and name of the procedure we performed that day. I initially listed the procedure for my main experiment, following space set aside for a table of contents.  

Results 

Despite the premature discontinuation of the experiment, I am proud to share that my agar plates were a success. Though new to me, the process of changing the tanks and feeding the Zebrafish was also carried out successfully. This first-time experience, without any prior knowledge, was a testament to the effectiveness of our approach, even in the face of unexpected circumstances. 

Discussion/Turn of Events 

In a significant turn of events, I have made the difficult decision to continue my academic journey at Vanderbilt University as a Biomedical Engineering student. This decision was not easily made, and it brings with it the uncertainty of being able to complete my experiment before my departure. However, I am certain that the lessons I have learned will undoubtedly shape my future endeavors.  

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Fluid-Structure Interaction of Flexible Thin Sheet

By Josiah Cassidy

 

Introduction

The environmental effect and limited supplies of fossil fuel energies have prompted extensive study into the creation of innovative and diversified methods to produce electrical energy. Parallel to this, systems capable of cheaply producing limited amounts of energy to power remote or isolated devices, for which connection to the standard electrical network is unfeasible due to cost or technical complexity, have also received special attention. These two factors have heightened interest in methods capable of producing self-sustaining vibrations of a solid or flexible substrate and converting the accompanying mechanical energy into electrical power. The conversion of kinetic energy from geophysical movements such as tidal currents, winds, and river flows into electricity is particularly interesting due to this energy source’s widespread availability and low environmental impact. Several mechanisms, such as vortex-induced vibration, flutter, and coupled- mode flutter instability of the flexible plate in a steady flow, have been identified through research on fluid–structure interactions (FSI) [1]. In this scenario, it is well known that the flat equilibrium state of the plate becomes unstable above a certain flow velocity, at which point significant amplitude dynamic vibrations can form on the structure. The goal of the proposed project is to investigate the capacity to generate electrical power from the self-sustained oscillations of a flexible plate caused by this fluttering instability by converting mechanical strain into electric potential using multiple embedded piezo electronic sensors.

Methods

Through a set of water tunnel experiments, we investigated the onset of flexible plate’s dynamic instability and the occurrence of possible limit cycle oscillations as a function of flow velocity and geometrical dimensions. We studied the effect of the geometrical parameters, such as the ratio between the length, width, and thickness of the plate, on the onset of the instabilities. The flexible plate was fabricated by pouring silicon rubber into 3D-printed molds to ensure both shape accuracy and structural flexibility. During this process, multiple piezoelectric sensors were embedded at various locations across the plate. The experiments for the dynamic response measurement were conducted in a re-circulating water tunnel facility at FSI LAB at UMass Dartmouth. To be able to conduct the experiment in our water tunnel, we have also designed a setup that will secure the flexible plate in the water tunnel shown in Figure 1.
Figure 1: Model of Flexible Plate Setup

The structural response of the system was measured using the embedded piezoelectric sensors purchased with OUR grant funds. Each piezoelectric sensor was connected using full bridge rectifiers to separate inputs of the Arduino in order to collect each sensor’s data separately but simultaneously. As the flexible plate starts to oscillate, it causes a strain in the sensor which transfers data to the Arduino in the form of voltage measurements based on the amount of strain experienced. Using this setup, we performed experiments with various sensors locations along the flexible plate. This method allowed us to find how the strain in the sensor changes based on not only the fluid flow and the flexible plate’s aspect ratio but also the geometric location of the piezoelectric sensor. Throughout this spring semester, many changes needed to be made in order for this setup to operate accurately and efficiently. First, new molds need to be designed and 3D printed so that we can do testing with many different aspect ratio flexible plates. We also needed to adjust the mounting setup for the flexible plate so that it would be fixed securely in the water tunnel. We made these adjustments by redesigning the holder in SOLIDWORKS to better support the flexible plate when oriented vertically. Other modifications were added like screws to secure the flexible plate in place during testing. Next, we adjusted our Arduino setup in order to accommodate 4 data inputs. This allows us to read data from multiple piezos on a single flexible plate during testing at the same time. A picture of the flexible plate during testing in the water tunnel is shown below in Figure 2.
Figure 2: Flexible Plate in Water Tunnel

Results

Figure 3 shows one of our flexible plates and an example of piezo placement in the plate. Some preliminary results are shown in Figure 4.
Figure 3: Placement of piezoelectric sensors in flexible plate
Figure 4: Graph comparing voltage output to water tunnel speed

The y-axis shows average voltage which was found from data collection through the use of the Arduino. This data was streamed to an excel file where it was averaged for each water tunnel speed test. Across the x-axis is the speed of the water flow in the water tunnel in meters per second. From this preliminary testing, we observed a higher voltage output from piezos placed toward the front of the flexible plate when compared to the rear. This could be due to the greater amount of change in displacement that occurs at these water tunnel speeds. A sample time series of the sensor measurements is shown below in Figure 5 in which you can see the changes in voltage output as the flexible plate experiences displacement.
Figure 5: “Sample Time Series of Sensor Data”

Conclusion and Future Direction

While our current testing has started to grant some results, much more testing is required before anything definitive can be said. As this project continues into the next semester or two, much more testing in the water tunnel should be done with more aspect ratios and a wider variety of piezo placement. Researchers should also use other methods like computer simulations to determine exactly why certain piezo placements give higher voltage outputs. This combined with our current piezo experiments may lead to more accurate predictions of power output during varied conditions without the need for experimental testing.

Reference

[1] A.K. Pandey, G. Sharma, R. Bhardwaj, Flow-induced reconfiguration and cross-flow vibrations of an elastic plate and implications to energy harvesting, Journal of Fluids and Structures 122 (October 2023), 103977. https://doi.org/10.1016/j.jfluidstructs.2023.103977.

RESEARCH IN BIOENGINEERING

Recreating a Recombinant R.opacus Bacteria that Can Use Chitin

By Jackie Ramirez

 

Introduction

In 2022, roughly 119 million pounds of American lobster (Homarus americanus) were landed, and this catch was valued at around $515 million. With this gigantic haul of seafood, consumers will eat <50% of the animal, which makes up the lobster meat. The majority of lobster biomass is inedible and is discarded by homes, restaurants and other facilities, and the majority of that waste is lobster shell. The lobster shell contains three main constituents: minerals like calcium carbonate (CaCO3), proteins and chitin/chitosan polysaccharide. Of these shell components, chitin and chitosan have shown value in bio-based processes. Chitin and chitosan are carbohydrate polymers consisting of the amino sugars N-acetyl-D-glucosamine (glcNAc) and/or D-glucosamine (glcN) monomer units. Depending on the degree of acetylation of the polysaccharide, the polymer may be called chitin or chitosan, where the majority of the monomer concentration of chitosan is D-glucosamine. Chitin and chitosan are very attractive biomaterials with a range of household and industrial uses. Regardless, there remains a large percentage of lobster shells that are discarded or underutilized.

Chitin as a biomaterial for biofuel production is a promising and new area of research that will contribute to solving the global climate crisis. Chitinase ChiA, ChiB, and ChiC break down chitin into monomers of N-acetyl glucosamine (NAG). ChiA is an endochitinase that breaks down chitin within a polymer. ChiB and ChiC are exo-chitinases that cleave monomers at the end of a polymer. The monomers produced by these enzymes are used to produce triacylglycerols (TAG). From here, the triacyclglycerols can be trans-esterified into biodiesel. The bioengineering department here at UMass Dartmouth has looked to the surrounding South Coast of Massachusetts as a source of chitin for biofuel production. The shells of crustaceans comprise of 40% chitin by weight. Through research efforts at UMass Dartmouth, chitin has been derived and separated from the protein components of lobster shells (1). This ecofriendly extraction method has given researchers here the ability to utilize crustacean waste from human consumption to isolate chitin and use it for biofuels in conjunction with Rhodococcus Opacus (R. Opacus). R. Opacus, strain PD630, is a gram-positive microbe which will accumulate TAG in the presence of a steady carbon source. It’s high lipid storage ability and rapid turnover rate make it an excellent candidate for biofuel production (2). Chitin is a proposed carbon source for the bacterium. R. Opacus, which is unable to produce the chitinases necessary to break down chitin into its monomer counterparts for biofuel production, therefore this project will make a recombinant strain of R. Opacus to express and secrete chitinase enzymes.

Soon after receiving an OUR grant, my mentor and her collaborators changed the strategy to use the shells. R.opacus is a difficult bacteria to genetically manipulate, so they decided to get another bacteria that is easier to manipulate and has a better chance of taking up plasmids that have the chitinase genes on them. We switched to pseudomonas aeruginosa.

Methods

Genomic DNA isolation using the Promega gDNA isolation kit.

Design primers specific for ChiA from the bacteria S. marcescens and amplify the gene from the genomic DNA.

Initial PCR conditions: Using 5ul gDNA, 1ul of ChiA-For primer and 1ul ChiA-rev primer plus Taq Supermix. The reaction proceeded with standard PCR cycle parameters with annealing at 59 degrees Celsius and 45 cycles.

Second Attempt using a temperature gradient to see what temperature is ideal for primers to anneal to the template.

Figure 1: We tested 57 degrees upto 62 degrees. Each bar indicates the temperature in that well.

Third attempt PCR: We switched to using Q5 high-fidelity Taq DNA polymerase.

Figure 2: These are the PCR Parameters we used with High Fidelity Taq.

Results

Initial attempts to amplify the ChiA, ChiB and ChiC genes from s.marcescens gDNA (Figure 3). The faint bands at the bottom of each lane are primer dimers. We are expecting bands between 1.0kb and 1.5kb. After some research we decided to try using a Taq polymerase that had High Fidelity. The reason was because we are trying to find one gene in a genome of 5,241,455 bp, and we figured that a DNA polymerase that could stay associated with the template better might allow us to get the genes. The High fidelity Q5 DNA polymerase resulted in the expected products between 1kb and 1.5 kb (Figure 4).

                        

Figures 3 (L) and 4 (R): First attempts to amplify ChiA, B and C. 

Having figured out how to get the correct bands I focused on Chitinase B. I was able to amplify ChiB and gel purify only the correct sized band (Figure 5).

Figure 5: Gel purified ChiB genes

The project is being continued by another student. The next steps are to cut the ChiB insert with enzymes and insert it into the vector.

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SKOV3 Ovarian Cancer Cells Research

By Ilya Korovaev

 

Abstract

Ovarian cancer stays undetected in 70% of cases until stages II, III, and 5-year survival rate is 36% for stage III, but this rate can have significant improvement if ovarian cancer could be detected at an early stage. It has been proven that cancerous cells actively produce exosomes, specifically SKOV3 ovarian cancer cells produce around 20000 exosomes per day and secrete them into the blood stream or lymph. If there will be a detection technique that could find those exosomes inside the body fluids, then ovarian cancer could be detected in the early stages. My research is focused on SKOV3 ovarian cancer cells culturing with the following exosome extraction and studying.

Introduction

Exosomes are nanoscaled extracellular vesicles secreted by cells with a size from 30 to 150nm. An exosome has phospholipids double-layer with specific protein markers on its surface and can contain DNA, RNA or proteins. Cancer cells use those exosomes to prepare other regions of the body for metastases acceptation. The process is the following: created in cancer cell exosome contains protein and DNA fragments to enter healthy cells and start the process of healthy cells mutation. Next step is to colonize prepared area with the metastases. The number of secreted exosomes by a single cancer cell is around 20000 per day, and they are getting secreted into lymph or blood flow to get to their destinations. SKOV3 ovarian cancer cells secrete exosomes with tetraspanins exosomal markers: CD9, CD63, CD81. CD9, CD8 which can be used to detect them among other exosomes and start the treatment as soon as the exosomes are detected. For easier exosomes detection they will be excreted from the cells and studied.

Methods

Cell Thawing:

Cells were taken from -80°C freezer and placed on the ice. After thawing process is completed, the cells were put into prewarmed cell PBS media at 37°C and centrifuged at 120RPM for 8 min. Pour the media out using a pipet and put 1 mL new media and resuspend the cells in the media using pipet. Transfer cells with media into culturing flask and add another 4 mL. Put culturing flask containing the cells and media into incubator at 37°C.

Media Change:

Remove the media from the flask using pipet and put 5 mL of fresh media into the flask. Put the flask with cells into the incubator at 37°C. Media change has been done every two days for the first culture and every three days for the second culture.

Cell Splitting:

Remove the media from the culturing flask using pipet. Put 1 mL of trypsin and rinse the culturing flask with it, put another 1 mL of trypsin and put the flask into the incubator for 10  minutes. Put media in the ratio 2:1 2- the media, 1 – trypsin. Centrifuge the solution for 6 min at 130RPM and pour the media out. Resuspend the cells in 1mL of media. Count the cells: put 10µL of the resuspended cells and 10 μL Trypan blue and pipet the solution onto counting plates on each side. Use cell counting machine and calculate the number of cells. If it is less than 1 million cell cells were put into culturing flask and follow media change process. If the number of cells exceeds 1 million cells cell can be transversed into 6-well plate 120 µL of cells will be transferred into 4 plates. Into first two wells 2ml of FBS media with exosomes will be added and 2mL of exosome-free FBS media. Next step is to monitor the growth of the cells to determine if exosomes-rich media speeds up the growth of the cells.

Results

The first culture of cells survived very well, and after 5 media changes it reached the 1million cells mark. After transferring those cells into 6-well plate on the second day cells got contaminated and died. The second culture of the cells I decided to change the media replacement. I changed the media every third day, and, after two media changes, the cells showed good growth but after the fourth media change, they all died. For the third and final time, I decided to keep media change on every second day because it showed the best results. After changing the media 4 times I decided to split them. Before splitting I prepared 4 different medias: normal (without synthetic FBS), synthetic (without normal FBS), 50% (with 0.5ml synthetic and 0.5 normal FBS), 25% (with 0.75mL synthetic and 0.25 mL normal FBS. After cell splitting and putting cells into 4 different plates and adding 2 mL of each media into four plates, cells died before next media change.

Conclusion

After running the cell culture for three times, all three times cells died. The first time it happened because of contamination; and the two other times, I assume that they did not have enough nutrients to survive.

 

References

  1. Siegel, R. L.; Miller, K. D.; Jemal, A., Cancer Statistics, 2019. CA: A Cancer Journal for Clinicians 2019, 69 (1), 7-34.
  2. Zhang, X.; Yuan, X.; Shi, H.; Wu, L.; Qian, H.; Xu, W., “Exosomes in Cancer: Small Particle, Big Player.”
    Journal of Hematology & Oncology 2015, 8 (1), 83.
  3. A. N. Böing, E. van der Pol, A. E. Grootemaat, F. A. W. Coumans, A. Sturk, and R. Nieuwland, “Single-step Isolation of Extracellular Vesicles by Size-exclusion Chromatography,” J Extracell Vesicles, vol. 3, no. 1, 2014, doi: 10.3402/jev.v3.23430.
  4. Nowak, M.; Janas, Ł.; Stachowiak, G.; Stetkiewicz, T.; Wilczyński, J. R., Current Clinical Application of Serum Biomarkers to Detect Ovarian Cancer. Przeglad menopauzalny = Menopause review 2015, 14 (4), 254-259
  5. Mashouri, Ladan, et al. “Exosomes: Composition, Biogenesis, and Mechanisms in Cancer Metastasis and Drug Resistance.” Molecular Cancer, vol. 18, no. 1, 2 Apr. 2019, https://doi.org/10.1186/s12943-019-0991-5.
  6. Tai, Yu‐Ling, et al. “Exosomes in Cancer Development and Clinical Applications.” Cancer Science, vol. 109, no. 8, 1 Aug. 2018, pp. 2364–2374, www.ncbi.nlm.nih.gov/pmc/articles/PMC1182327. https://doi.org/10.1111/cas.13697. Accessed 27 May 2020.
  7. Raposo G, Stoorvogel W. “Extracellular Vesicles: Exosomes, Microvesicles, and Friends.” J Cell Biol. Feb 18, 2013; 200 (4): 373-83. doi: 10.1083/jcb.201211138. PMID: 23420871; PMCID: PMC3575529.

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Presentation at the 76th Annual Meeting of the Division of Fluid Dynamics in Washington D.C., November 19-21, 2023.

By Jordan I Breveleri

I attended the 76th Annual Meeting of the APS Division of Fluid Dynamics in Washington D.C. from November 19-21, 2023, where I presented my research on drag reduction in marine vessels using porous superhydrophobic surfaces (SHS). The conference served as a dynamic platform for researchers to exchange ideas and advancements in the field. The APS (American Physical Society) Division of Fluid Dynamics Conference provided an excellent platform for researchers to discuss and share their findings in fluid dynamics.

My presentation focused on the innovative use of porous SHS to reduce drag in marine vessels. By injecting gas through the porous surface, an air layer can be sustained, effectively minimizing drag. The presentation primarily showcased the results of my research and highlighted its potential applications in fluid dynamics. The surreal atmosphere of Washington D.C., steeped in history, offered an inspiring setting for scientific discourse. The city’s rich cultural and national significance added an extra layer of depth to the conference experience, making it both professionally and personally enriching.

Photo of Jordan I Breveleri in Washington D.C. 

Presenting in front of a diverse audience was initially nerve-wracking, but with the support of my professor and peers, I successfully navigated the challenge. The engaging discussions and feedback further enhanced the presentation experience, providing valuable insights into my work. Aside from presenting my research, I attended various talks during the conference, gaining diverse insights into fluid dynamics. One particularly intriguing presentation focused on aurora lights, offering a fascinating perspective on the broader spectrum of research within the field.

The conference was an invaluable experience. Presenting my research and attending other panels created a memorable professional journey. The conference not only provided a platform for knowledge exchange but also fostered connections and collaboration within the fluid dynamics community. Overall, it was a rewarding experience that contributed significantly to my understanding of the field.

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IMECE Undergraduate Student Poster Competition 2023

By Chloe Shirikjian

 

The International Mechanical Engineering Congress and Exposition or IMECE Undergraduate Student Poster Competition and Conference was an impactful event that enabled me to present my research to peers, form connections with people in research and industry worldwide, and to learn about cutting-edge technologies. I arrived in New Orleans on Sunday, October 29, 2023, and attended an orientation for first-time conference attendees. Important members of ASME (American Society of Mechanical Engineers) welcomed the students and shared information about the conference and volunteering opportunities.

I presented my work at the Undergraduate Student Poster Competition later that day. During the first hour of the competition, I presented my poster in front of numerous judges. My poster titled, An Integrated Computational Framework for Process-informed Analysis of 3D Printed Knee Assembly Components, displayed my research from the past six months on numerical simulations of additive manufacturing (AM). I first informed attendees about the background and project goals. Then, I spoke in detail about the application of additive manufacturing to patient-specific prosthesis design and how my objectives contribute to that goal. I went on to discuss the setup, results, and conclusion. In summary, the residual stresses present in the AM printed parts will be a determining factor for structural failure. Additionally, computational methods for function-oriented tolerancing must be developed for practical application of AM in the industry. This event allowed me to receive feedback from judges on points that I had not yet considered, including displaying my results in a xy-plot and including more realistic parameters, such as ligaments and tendons, into my simulation. Furthermore, I received encouraging feedback about the need for this type of work in the industry and received compliments about my presentation. Engaging with judges and peers sparked interesting discussions and a new passion for continuing my research.

 

 

Chloe Shirikjian next to her poster at the IMECE Undergraduate Student Poster Competition and Conference 2023

 

The next day, I attended a talk from a keynote speaker and multiple technical sessions. This event allowed me to learn about the cutting-edge research currently being done and explore various interesting topics in mechanical engineering. In particular, the keynote speaker presented on Small Satellites and the Future of Planetary Space Exploration, which discussed Georgia Tech’s accomplishment of being the first university to send a small satellite into space. The mission was originally intended to use the satellite to search for ice on areas of the moon that do not receive sunlight. However, they experienced some issues with one of the satellite thrusters, which sent the satellite off its original course. This talk enlightened me on the newfound ability for private industry space exploration. In the 2020s, private companies can explore space on a national scale with small satellites and rideshares; space exploration is no longer limited to government defense companies.

My experience at IMECE allowed me to form connections and to reignite my passion for engineering. Presenting my work allowed me to see my research from different perspectives and connect with people in the industry interested in additive manufacturing. Furthermore, attending the technical sessions allowed me to learn about new technologies in industry and research fields. I am extremely grateful to experience such an extraordinary event and I look forward to pursuing similar opportunities in the future.